Alpha1beta2gamma2 GABA Research Articles

Carisoprodol-Mediated Modulation of GABAA Receptors: In Vitro and in Vivo Studies

Carisoprodol is a frequently prescribed muscle relaxant. In recent years, this drug has been increasingly abused. The effects of carisoprodol have been attributed to its metabolite, meprobamate, a controlled substance that produces sedation via GABA(A) receptors (GABA(A)Rs). Given the structural similarities between carisoprodol and meprobamate, we used electrophysiological and behavioral approaches to investigate whether carisoprodol directly affects GABA(A)R function. In whole-cell patch-clamp studies, carisoprodol allosterically modulated and directly activated human alpha1beta2gamma2 GABA(A)R function in a barbiturate-like manner. At millimolar concentrations, inhibitory effects were apparent. Similar allosteric effects were not observed for homomeric rho1 GABA or glycine alpha1 receptors. In the absence of GABA, carisoprodol produced picrotoxin-sensitive, inward currents that were significantly larger than those produced by meprobamate, suggesting carisoprodol may directly produce GABAergic effects in vivo. When administered to mice via intraperitoneal or oral routes, carisoprodol elicited locomotor depression within 8 to 12 min after injection. Intraperitoneal administration of meprobamate depressed locomotor activity in the same time frame. In drug discrimination studies with carisoprodol-trained rats, the GABAergic ligands pentobarbital, chlordiazepoxide, and meprobamate each substituted for carisoprodol in a dose-dependent manner. In accordance with findings in vitro, the discriminative stimulus effects of carisoprodol were antagonized by a barbiturate antagonist, bemegride, but not by the benzodiazepine site antagonist, flumazenil. The results of our studies in vivo and in vitro collectively suggest the barbiturate-like effects of carisoprodol may not be due solely to its metabolite, meprobamate. Furthermore, the functional traits we have identified probably contribute to the abuse potential of carisoprodol.

The Anesthetic-Like Effects of Diverse Compounds on Wild-Type and Mutant γ-Aminobutyric Acid Type A and Glycine Receptors

No theory of inhaled anesthetic action requires volatility of the anesthetic to accomplish the biophysical interaction of anesthetic with biological target. The identification of mutations that attenuate the effect of inhaled anesthetics on various receptors raises the possibility that nonvolatile compounds with anesthetic effects can be identified with the aid of these receptors. In previous studies, we identified compounds that were either charged or had an exceptionally low vapor pressure and which modulated anesthetic-sensitive receptors in a manner similar to inhaled anesthetics. We tested whether these, and another charged compound, shared a common mechanism with volatile anesthetics, by comparing their effect on wild-type gamma-aminobutyric acid type A (GABA(A)) or glycine receptors and mutant receptors that were engineered to be relatively resistant to inhaled anesthetics. The effect of beta-hydroxybutyric acid, ammonium chloride, diethylhexyl phthalate, and GABA were tested on homomeric alpha1 and mutant alpha1 (S267I) glycine receptors. The effect of sodium dodecyl sulfate and glycine were tested on alpha1 b2 gamma2s and mutant alpha1(S270I) beta2 gamma2s GABA(A) receptors. Receptors were expressed in Xenopus laevis oocytes and studied using two-electrode voltage clamping. For both GABA(A) and glycine receptors, isoflurane and ethanol were used as positive controls and propofol as a negative control (i.e., unaffected by the mutation). Beta-hydroxybutyric acid, ammonium chloride, diethylhexyl phthalate, and GABA all enhanced glycine receptor function. This effect was reduced by the S267I mutations. Sodium dodecyl sulfate and glycine enhanced GABA(A) receptor function, and the S270I mutation attenuated this effect. These findings support the hypothesis that the compounds studied modulate GABA(A) or glycine receptors by a mechanism similar to that of isoflurane and ethanol. Comparing the effect of drugs on anesthetic-sensitive wild-type receptors with relatively less sensitive mutant receptors may help identify compounds with anesthetic effects.

An Updated Unified Pharmacophore Model of the Benzodiazepine Binding Site on γ-Aminobutyric Acida Receptors: Correlation with Comparative Models

A successful unified pharmacophore/receptor model which has guided the synthesis of subtype selective compounds is reviewed in light of recent developments both in ligand synthesis and structural studies of the binding site itself. The evaluation of experimental data in combination with a comparative model of the alpha1beta2gamma2 GABA(A) receptor leads to an orientation of the pharmacophore model within the Bz BS. Results not only are important for the rational design of selective ligands, but also for the identification and evaluation of possible roles which specific residues may have within the benzodiazepine binding pocket.

Identification of Anesthetic Binding Sites on Human Serum Albumin Using a Novel Etomidate Photolabel

We have synthesized a novel analog of the general anesthetic etomidate in which the ethoxy group has been replaced by an azide group, and which can be used as a photolabel to identify etomidate binding sites. This acyl azide analog is a potent general anesthetic in both rats and tadpoles and, as with etomidate, is stereoselective in its actions, with the R(+) enantiomer being significantly more potent than the S(-) enantiomer. Its effects on alpha1beta2gamma2s GABA(A) receptors expressed in HEK-293 cells are virtually indistinguishable from the parent compound etomidate, showing stereoselective potentiation of GABA-induced currents, as well as direct mimetic effects at higher concentrations. In addition, a point mutation (beta2 N265M), which is known to attenuate the potentiating actions of etomidate, also blocks the effects of the acyl azide analog. We have investigated the utility of the analog to identify etomidate binding sites by using it to photolabel human serum albumin, a protein that binds approximately 75% of etomidate in human plasma and which is thought to play a major role in its pharmacokinetics. Using HPLC/mass spectrometry we have identified two anesthetic binding sites on HSA. One site is the well-characterized drug binding site I, located in HSA subdomain IIA, and the second site is also an established drug binding site located in subdomain IIIB, which also binds propofol. The acyl azide etomidate may prove to be a useful new photolabel to identify anesthetic binding sites on the GABA(A) receptor or other putative targets.

Allosteric uncoupling and up-regulation of benzodiazepine and GABA recognition sites following chronic diazepam treatment of HEK 293 cells stably transfected with α1β2γ2S subunits of GABAA receptors

Benzodiazepines are drugs known to produce tolerance and dependence and also to be abused and co-abused. The aim of this study was to further explore the mechanisms that underlie adaptive changes in GABA(A) receptors following prolonged exposure to these drugs. Human embryonic kidney (HEK 293) cells stably expressing recombinant alpha1beta2gamma2s GABA(A) receptors were exposed for 72 h to a high concentration of diazepam (50 microM) in the absence or presence of other drugs. Radioligand binding studies were used to determine the parameters of [(3)H]flunitrazepam and [(3)H]muscimol binding sites and allosteric interactions between these sites. Prolonged treatment with diazepam increased the maximum number (B (max)) of [(3)H]flunitrazepam and [(3)H]muscimol binding sites in the membranes, and of [(3)H]muscimol binding sites on the surface of HEK 293 cells. There was no change in the affinity (K (d)) of binding sites. The diazepam-induced increase in the B (max) value of [(3)H]flunitrazepam binding sites was reduced by two GABA(A) receptor antagonists, gabazine (1 and 10 microM) and picrotoxin (100 microM). In addition, it was reduced by cycloheximide (5 microg/ml), a protein synthesis inhibitor, and actinomycin D (7.5 microg/ml), an RNA synthesis inhibitor. Flumazenil (5 microM), the antagonist of benzodiazepine binding sites, also up-regulated [(3)H]flunitrazepam recognition sites. Simultaneous treatment with diazepam and flumazenil failed to produce an additive up-regulation. GABA (1 nM - 1 mM)-induced potentiation of [(3)H]flunitrazepam binding to membranes obtained from diazepam (50 microM)-pretreated cells was markedly reduced, suggesting functional uncoupling between GABA and benzodiazepine binding sites. The results suggest that diazepam up-regulated benzodiazepine binding sites on stably expressed GABA(A) receptors by stimulating their synthesis at both the transcriptional and translational levels. A comparable increase of [(3)H]muscimol binding sites expressed on the surface of intact HEK 293 cells suggests that internalisation of surface receptors presumably can not explain the uncoupling.

Mechanism of action of benzodiazepines on GABAA receptors

Wild-type and mutant alpha1beta2gamma2 GABA(A) receptors were expressed in Xenopus laevis oocytes and examined using the two-electrode voltage clamp. Dose-response relationships for GABA were compared in the absence and presence of 1 microM diazepam (DZP) or methyl-6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate (DMCM). The dose-current relationships yielded EC(50)'s (concentration for half-maximal activation) of 41.0+/-3.0, 21.7+/-2.7, and 118.3+/-6.8 microM for GABA, GABA plus DZP, and GABA plus DMCM, respectively.DZP- and DMCM-mediated modulation were examined in GABA(A) receptors in which the beta-subunit carries the L259S mutation. This mutation has been shown to produce spontaneous opening and impart a leftward shift in the dose-response relationship. In this case, neither DZP nor DMCM produced a significant alteration in the GABA dose-response relationship with GABA EC(50)'s of 0.078+/-0.005, 0.12+/-0.03, and 0.14+/-0.004 microM for GABA, GABA plus 1 microM DZP, and GABA plus 1 microM DMCM.DZP- and DMCM-mediated modulations were examined in GABA(A) receptors in which the alpha-subunit carries the L263S mutation. This mutation also produced spontaneous opening and a leftward shift of the GABA dose-response relation, but to a lesser extent than that of betaL259S. In this case, the leftward and rightward shifts for DZP and DMCM were still present with EC(50)'s=0.24+/-0.03, 0.14+/-0.02, and 1.2+/-0.04 microM for GABA, GABA plus 1 microM DZP, and GABA plus 1 microM DMCM, respectively.Oocytes expressing ultrahigh levels of wild-type GABA(A) receptors exhibited currents in response to 1 muM DZP alone, whereas DMCM decreased the baseline current. The DZP-mediated activation currents were determined in wild-type receptors as well as receptors in which the GABA binding site was mutated (beta2Y205S). The EC(50)'s for DZP-mediated activation were 72.0+/-2.0 and 115+/-6.2 nM, respectively, similar to the EC(50) for DZP-mediated enhancement of the wild-type GABA-activated current (64.8+/-3.7 nM). Our results support a mechanism in which DZP increases the apparent affinity of the receptor, not by altering the affinity of the closed state, but rather by shifting the equilibrium towards the high-affinity open state.

The Pheromone Androstenol (5α-Androst-16-en-3α-ol) Is a Neurosteroid Positive Modulator of GABAA Receptors

Androstenol is a steroidal compound belonging to the group of odorous 16-androstenes, first isolated from boar testes and also found in humans. Androstenol has pheromone-like properties in both animals and humans, but the molecular targets of its pheromonal activity are unknown. Androstenol is structurally similar to endogenous A-ring reduced neurosteroids that act as positive modulators of GABA(A) receptors. Here we show that androstenol has neurosteroid-like activity as a GABA(A) receptor modulator. In whole-cell recordings from cerebellar granule cells, androstenol (but not its 3beta-epimer) caused a concentration-dependent enhancement of GABA-activated currents (EC(50), 0.4 microM in cultures; 1.4 microM in slices) and prolonged the duration of spontaneous and miniature inhibitory postsynaptic currents. Androstenol (0.1-1 microM) also potentiated the amplitude of GABA-activated currents in human embryonic kidney 293 cells transfected with recombinant alpha1beta2gamma2 and alpha2beta2gamma2 GABA(A) receptors and, at high concentrations (10-300 microM), directly activated currents in these cells. Systemic administration of androstenol (30-50 mg/kg) caused anxiolytic-like effects in mice in the open-field test and elevated zero-maze and antidepressant-like effects in the forced swim test (5-10 mg/kg). Androstenol, but not its 3beta-epimer, conferred seizure protection in the 6-Hz electroshock and pentylenetetrazol models (ED(50) values, 21.9 and 48.9 mg/kg, respectively). The various actions of androstenol in the whole-animal models are consistent with its activity as a GABA(A) receptor modulator. GABA(A) receptors could represent a target for androstenol as a pheromone, for which it is well suited because of high volatility and lipophilicity, or as a conventional hormonal neurosteroid.

Activation of single heteromeric GABAA receptor ion channels by full and partial agonists

The linkage between agonist binding and the activation of a GABA(A) receptor ion channel is yet to be resolved. This aspect was examined on human recombinant alpha1beta2gamma2S GABA(A) receptors expressed in human embryonic kidney cells using the following series of receptor agonists: GABA, isoguvacine, 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP), isonipecotic acid, piperidine-4-sulphonic acid (P4S), imidazole-4-acetic acid (IAA), 5-(4-piperidyl)-3-isothiazolol (thio-4-PIOL) and 5-(4-piperidyl)-3-isoxazolol (4-PIOL). Whole-cell concentration-response curves enabled the agonists to be categorized into four classes based upon their maximum responses. Single channel analyses revealed that the channel conductance of 25-27 pS was unaffected by the agonists. However, two open states were resolved from the open period distributions with mean open times reduced 5-fold by the weakest partial agonists. Using saturating agonist concentrations, estimates of the channel shutting rate, alpha, ranged from 200 to 600 s(-1). The shut period distributions were described by three or four components and for the weakest partial agonists, the interburst shut periods increased whilst the mean burst durations and longest burst lengths were reduced relative to the full agonists. From the burst analyses, the opening rates for channel activation, beta, and the total dissociation rates, k(-1), for the agonists leaving the receptor were estimated. The agonist efficacies were larger for the full agonists (E approximately 7-9) compared to the weak partial agonists ( approximately 0.4-0.6). Overall, changes in agonist efficacy largely determined the different agonist profiles with contributions from the agonist affinities and the degree of receptor desensitization. From this we conclude that GABA(A) receptor activation does not occur in a switch-like manner since the agonist recognition sites are flexible, accommodating diverse agonist structures which differentially influence the opening and shutting rates of the ion channel.

Desensitization Mechanism of GABA Receptors Revealed by Single Oocyte Binding and Receptor Function

Prolonged exposure of most fast neurotransmitter-operated ion channels to agonist drives the receptors into a nonfunctional, or desensitized, state. Despite extensive investigation, desensitization remains a thoroughly characterized, yet poorly understood, process. Part of the difficulty in elucidating the mechanism of desensitization has been an inability to resolve the kinetics of both agonist binding and functional desensitization in the same set of operable receptors. To overcome this limitation, we applied single oocyte 3H-ligand binding and two-electrode voltage clamp to oocytes expressing recombinant alpha1beta2gamma2 GABA receptors. Using this approach, we report several observations fundamental to the mechanism of desensitization. First, we confirm that desensitization reversibly shifts GABA receptors into a high-affinity state. For [3H]GABA binding, the half-maximal binding of the desensitized state was approximately 0.040 microm. Second, we show that, upon agonist removal, this high-affinity state disappears with a time constant of 127 +/- 12 sec (n = 4), similar to the time constant for functional recovery from desensitization of 124 +/- 26 sec (n = 5). [3H]GABA, however, dissociates fourfold faster (tau = 30 +/- 2 sec; n = 3) than functional recovery, indicating that desensitized receptors need not be bound by GABA. These data provide direct evidence for a cyclical model of receptor desensitization.

Two Different Mechanisms of Disinhibition Produced by GABAAReceptor Mutations Linked to Epilepsy in Humans

The first mutations of the GABA(A) receptor channel linked to familial epilepsy in humans were reported recently (Baulac et al., 2001; Wallace et al., 2001). Preliminary functional analysis of alpha1beta2gamma2 GABA(A) receptors expressed in Xenopus oocytes suggested that the gamma2 subunit R43Q mutation abolished current enhancement by the benzodiazepine, diazepam, and that the gamma2 subunit K289M mutation decreased current amplitudes. We used single-channel recording and concentration jump techniques applied to outside out patches to evaluate the impact of these mutations on GABA(A) receptor channel function of the highly conserved rat ortholog subunits expressed in human embryonic kidney cells. When coexpressed with alpha1 and beta3 subunits, no differences were observed between wild-type and mutant GABA(A) receptor current activation rates or rates or extent of desensitization during prolonged (400 msec) GABA application (1 mm). Although deactivation after brief (5 msec) or prolonged (400 msec) GABA application was unaltered by the R43Q mutation, deactivation (a correlate of IPSC duration) was accelerated for the K289M mutation. Faster deactivation was likely a consequence of altered gating, because single-channel openings had shorter mean duration. Interestingly, the R43Q mutation did not alter diazepam potentiation. It did, however, substantially decrease current amplitude, which was not caused by decreased single-channel conductance or open time, suggesting reduced surface expression of functional receptors. The two gamma2 subunit mutations likely produce disinhibition and familial epilepsy by distinct mechanisms, suggesting that maintenance of neuronal inhibition depends not only on the peak amplitude of IPSCs, but also on their time course.

Two different mechanisms of disinhibition produced by GABAA receptor mutations linked to epilepsy in humans.

The first mutations of the GABA(A) receptor channel linked to familial epilepsy in humans were reported recently (Baulac et al., 2001; Wallace et al., 2001). Preliminary functional analysis of alpha1beta2gamma2 GABA(A) receptors expressed in Xenopus oocytes suggested that the gamma2 subunit R43Q mutation abolished current enhancement by the benzodiazepine, diazepam, and that the gamma2 subunit K289M mutation decreased current amplitudes. We used single-channel recording and concentration jump techniques applied to outside out patches to evaluate the impact of these mutations on GABA(A) receptor channel function of the highly conserved rat ortholog subunits expressed in human embryonic kidney cells. When coexpressed with alpha1 and beta3 subunits, no differences were observed between wild-type and mutant GABA(A) receptor current activation rates or rates or extent of desensitization during prolonged (400 msec) GABA application (1 mm). Although deactivation after brief (5 msec) or prolonged (400 msec) GABA application was unaltered by the R43Q mutation, deactivation (a correlate of IPSC duration) was accelerated for the K289M mutation. Faster deactivation was likely a consequence of altered gating, because single-channel openings had shorter mean duration. Interestingly, the R43Q mutation did not alter diazepam potentiation. It did, however, substantially decrease current amplitude, which was not caused by decreased single-channel conductance or open time, suggesting reduced surface expression of functional receptors. The two gamma2 subunit mutations likely produce disinhibition and familial epilepsy by distinct mechanisms, suggesting that maintenance of neuronal inhibition depends not only on the peak amplitude of IPSCs, but also on their time course.

N-Substituted 4-amino-3,3-dipropyl-2(3H)-furanones: new positive allosteric modulators of the GABA(A) receptor sharing electrophysiological properties with the anticonvulsant loreclezole.

1,4-Addition of benzylamine to 2(5H)-furanone followed by dialkylation of the 3-position with allylbromide gave (+/-)-4-benzyl-3,3-diallyl-2(3H)-furanone (8), which served as the intermediate for the synthesis of various N-substituted 4-amino-3,3-dipropyl-2(3H)-furanones (+/-)-9a-l. The compounds were evaluated for their capacity to potentiate or inhibit GABA-evoked currents in Xenopus laevis oocytes expressing recombinant alpha1beta2gamma2 GABA(A) receptors. The benzyl, ethyl, and allyl carbamates ((R)-9a (100 microM), (+/-)-9b (100 microM), (+/-)-9c (200 microM)) stimulated GABA currents by 279 +/- 47%, 426 +/- 8%. and 765 +/- 61%, respectively, while the phenylcarboxamide (+/-)-9f (200 microM) stimulated currents by 420 +/- 33%. Concentration-response studies showed that compound 9c was approximately twice as potent in stimulating GABA currents as alpha-EMTBL (2), the most potent 3,3-dialkylbutyrolactone known to date. On the other hand, the N-sulfonyl analogues were much less active or even inhibited GABA-evoked currents. In vitro radioligand displacement studies on rat brain membranes showed that these compounds did not bind to the benzodiazepine or GABA recognition sites of the GABA(A) receptor. However, these compounds generally weakly displaced [(35)S]-TBPS (approximately 50% displacement at 100 microM), though potencies did not correlate with GABA current potentiation. Results obtained with alpha1beta1 and mutant alpha1beta2N265S receptors, which compared to alpha1beta2 receptors are both much less sensitive to current stimulation produced by the anticonvulsant loreclezole, suggest that at least some of these aminobutyrolactones, (e.g., 9a, 9c), and interestingly also alpha-EMTBL, share stimulatory properties with loreclezole.

Positive allosteric modulation by ultraviolet irradiation on GABA(A), but not GABA(C), receptors expressed in Xenopus oocytes.

1. Recombinant rat GABA(A) (alpha1beta2, alpha1beta2gamma2, beta2gamma2) and human GABA(C) (rho1) receptors were expressed in Xenopus oocytes to examine the effect of ultraviolet (UV) light on receptor function. 2. GABA-induced currents in individual oocytes expressing GABA receptors were tested by two-electrode voltage clamp before, and immediately after, 312 nm UV irradiation. 3. UV irradiation significantly potentiated 10 microM GABA-induced currents in alpha1beta2gamma2 GABA receptors. The modulation was irradiation dose dependent, with a maximum potentiation of more than 3-fold. 4. The potentiation was partially reversible and decayed exponentially with a time constant of 8.2 +/- 1.2 min toward a steady-state level which was still significantly elevated (2.7 +/- 0.3-fold) compared to the control level. 5. The effect of UV irradiation on GABA(A) receptors varied with receptor subunit composition. UV irradiation decreased the EC50 of the alpha1beta2, alpha1beta2gamma2 and beta2gamma2 GABA(A) receptors, but exhibited no significant effect on the rho1 GABA(C) receptor. 6. UV irradiation also significantly increased the maximum current 2-fold in alpha1beta2 GABA(A) receptors with little effect on the maximum of alpha1beta2gamma2 (1.1-fold) or beta2gamma2 (1.1-fold) GABA(A) receptors. 7. The effect of UV irradiation on GABA(A) receptors did not overlap the effect of the GABA receptor- allosteric modulator, diazepam. 8. The UV effect on GABA(A) receptors was not prevented by the treatment of the oocytes before and during UV irradiation with one of the following free-radical scavengers: 40 mM D-mannitol, 40 mM imidazole or 40 mM sodium azide. In addition, the effect was not mimicked by the free-radical generator, H2O2. 9. Potential significance and mechanism(s) of the UV effect on GABA receptors are discussed.

Effects of GABA(A) receptor partial agonists in primary cultures of cerebellar granule neurons and cerebral cortical neurons reflect different receptor subunit compositions.

Based on an unexpected high maximum response to piperidine-4-sulphonic acid (P4S) at human alpha1alpha6beta2gamma2 GABA(A) receptors expressed in Xenopus oocytes attempts to correlate this finding with the pharmacological profile of P4S and other GABA(A) receptor ligands in neuronal cultures from rat cerebellar granule cells and rat cerebral cortex were carried out. GABA and isoguvacine acted as full and piperidine-4-sulphonic acid (P4S) as partial agonists, respectively, at alpha1beta2gamma2, alpha6beta2gamma2 and alpha1alpha6beta2gamma2 GABA receptors expressed in Xenopus oocytes with differences in potency. Whole-cell patch-clamp recordings were used to investigate the pharmacological profile of the partial GABA(A) receptor agonists 4,5,6,7-tetrahydroisoxazolo-(5,4-c)pyridin-3-ol (THIP), P4S, 5-(4-piperidyl)isoxazol-3-ol (4-PIOL), and 3-(4-piperidyl)isoxazol-5-ol (iso-4-PIOL), and the competitive GABA(A) receptor antagonists Bicuculline Methbromide (BMB) and 2-(3-carboxypropyl)-3-amino-6-methoxyphenyl-pyridazinium bromide (SR95531) on cerebral cortical and cerebellar granule neurons. In agreement with findings in oocytes, GABA, isoguvacine and P4S showed similar pharmacological profiles in cultured cortical and cerebellar neurones, which are known to express mainly alpha1, alpha2, alpha3, and alpha5 containing receptors and alpha1, alpha6 and alpha1alpha6 containing receptors, respectively. 4-PIOL and iso-4-PIOL, which at GABA(A) receptors expressed in oocytes were weak antagonists, showed cell type dependent potency as inhibitors of GABA mediated responses. Thus, 4-PIOL was slightly more potent at cortical neurones than at granule neurones and iso-4-PIOL was more potent in inhibiting isoguvacine-evoked currents at cortical than at granule neurons. Furthermore the maximum response to 4-PIOL corresponded to that of a partial agonist, whereas that of iso-4-PIOL gave a maximum response close to zero. It is concluded that the pharmacological profile of partial agonists is highly dependent on the receptor composition, and that small structural changes of a ligand can alter the selectivity towards different subunit compositions. Moreover, this study shows that pharmacological actions determined in oocytes are generally in agreement with data obtained from cultured neurons.

A novel positive allosteric modulator of the GABA(A) receptor: the action of (+)-ROD188.

(+)-ROD188 was synthesized in the search for novel ligands of the GABA binding site. It shares some structural similarity with bicuculline. (+)-ROD188 failed to displace [(3)H]-muscimol in binding studies and failed to induce channel opening in recombinant rat alpha1beta2gamma2 GABA(A) receptors functionally expressed in Xenopus oocytes. (+)-ROD188 allosterically stimulated GABA induced currents. Displacement of [(3)H]-Ro15-1788 indicated a low affinity action at the benzodiazepine binding site. In functional studies, stimulation by (+)-ROD188 was little sensitive to the presence of 1 microM of the benzodiazepine antagonist Ro 15-1788, and (+)-ROD188 also stimulated currents mediated by alpha1beta2, indicating a major mechanism of action different from that of benzodiazepines. Allosteric stimulation by (+)-ROD188 was similar in alpha1beta2N265S as in unmutated alpha1beta2, while that by loreclezole was strongly reduced. (+)-ROD188 also strongly stimulated currents elicited by either pentobarbital or 5alpha-pregnan-3alpha-ol-20-one (3alpha-OH-DHP), in line with a mode of action different from that of barbiturates or neurosteroids as channel agonists. Stimulation by (+)-ROD188 was largest in alpha6beta2gamma2 (alpha6beta2gamma2>>alpha1beta2gamma2=alpha5beta2gamma2++ +>alpha2beta2ga mma2= alpha3beta2gamma2), indicating a unique subunit isoform specificity. Miniature inhibitory postsynaptic currents (mIPSC) in cultures of rat hippocampal neurons, caused by spontaneous release of GABA showed a prolonged decay time in the presence of 30 microM (+)-ROD188, indicating an enhanced synaptic inhibitory transmission.

Differential Sensitivity of Recombinant GABAA Receptors Expressed in Xenopus Oocytes to Modulation by Topiramate

This study evaluated the modulatory effects of topiramate (TPM) on various subtypes of recombinant rat gamma-aminobutyric acid A (GABA(A)) receptors expressed in Xenopus oocytes. Specific subunits of GABA(A) receptors were expressed in Xenopus oocytes. Voltage-clamp recordings of currents were performed after application of TPM (1-100 microM) to these oocytes in the presence or absence of GABA. In a concentration-dependent fashion, TPM (1-100 microM) reversibly inhibited GABA-evoked Cl- currents in oocytes expressing either alpha1beta2gamma2S and alpha2beta2gamma2S recombinant GABA(A) receptors and reduced the current-fading rate in alpha1beta2gamma2S-expressing oocytes. Topiramate was effective at GABA concentrations of 1-10 microM but not at 100 microM. Topiramate (1-100 microM) potentiated GABA-evoked Cl- currents and increased the fading rate in oocytes expressing the alpha6beta2gamma2S GABA(A) receptor. It had no effect on Cl- currents mediated through the alpha4beta2gamma2S receptor or through the mixed population of GABA(A) receptors expressed from rat brain mRNA. In general, the observed effects of TPM were more pronounced on fading rates than on peak Cl- currents. These results indicate that TPM may affect desensitization of GABA(A) receptors as assessed by changes in the fading rates of GABA-evoked Cl- currents, possibly by effects on second-messenger systems.

Dual mode of stimulation by the beta-carboline ZK 91085 of recombinant GABA(A) receptor currents: molecular determinants affecting its action.

In electrophysiological measurements the beta-carboline ethyl 6-benzyloxy-beta-carboline-3-carboxylate (ZK 91085) acts as a positive allosteric modulator on rat recombinant alpha1beta2gamma2 GABA(A) receptors and binds with high affinity (IC50-1.5 nM) to the [3H]-flunitrazepam site. Flumazenil was able to partially counteract the current modulation. These observations indicate an action of ZK 91085 at the benzodiazepine binding site. At the dual subunit combination alpha1beta2, which lacks the gamma subunit required for benzodiazepine modulation, we still observed a potentiation of GABA currents. Thus ZK 91085 acts via an additional site on the channel. At the subunit combination alpha1beta1, ZK 91085 potentiation is strongly reduced as compared to alpha1beta2. In binding studies, ZK 91085 was able to decrease [35S]-TBPS binding in alpha1beta2gamma2 and alpha1beta2 but not in alpha1beta1. This selectivity of ZK 91085 for receptors containing the beta2 isoform over those containing the beta1 isoform is reminiscent of the action of loreclezole. To identify amino acid residues important for the second type of modulation, we functionally compared wild type alpha1beta2 and mutant receptors for stimulation by ZK 91085. The mutation beta2N265S, that abolishes loreclezole effects, also abolishes ZK 91085 stimulation. The mutation beta2Y62L increased stimulation by ZK 91085 3-4 fold, locating an influencing entity of the second type of action of ZK 91085 at an alpha/beta subunit interface. Structural intermediates of ZK 91085 and the beta-carboline abecarnil, the latter of which only slightly potentiated GABA currents in alpha1/beta2, were analysed to determine structural requirements for modulation. ZK 91085 thus allosterically stimulates the GABA(A) receptor through two sites of action: the benzodiazepine site and the loreclezole site in contrast to classical beta-carbolines, that confer negative allosteric modulation through the benzodiazepine site.

2-Phenyl-imidazo[1,2-a]pyridine derivatives as ligands for peripheral benzodiazepine receptors: stimulation of neurosteroid synthesis and anticonflict action in rats.

Selective activation of peripheral benzodiazepine receptors (PBRs) in adrenal cells and brain oligodendrocytes promotes steroidogenesis. Three 2-phenyl-imidazo[1,2-a]pyridine derivatives (CB 34, CB 50 and CB 54) have now been investigated with regard to their selectivity for PBRs and their ability to stimulate central and peripheral steroidogenesis in rats. The three CB compounds (10(-10)-10(-4) M) potently inhibited the binding of the PBR ligand [3H]-PK 11195 to brain and ovary membranes in vitro, without substantially affecting [3H]-flunitrazepam binding to central benzodiazepine receptors. These compounds (10(-7)-10(-4) M) also had little or no marked effects on GABA-evoked Cl- currents in voltage-clamped Xenopus oocytes expressing human alpha1beta2gamma2S GABA(A) receptors. In addition, they failed to affect ligands binding to GABA(B), D1/D2 dopamine, muscarinic acetylcholine, N-methyl-D-aspartic acid and opiate receptors. Intraperitoneal administration of CB compounds (3-50 mg kg(-1)) induced a dose-dependent increase in the concentrations of neuroactive steroids in plasma and brain. The brain concentrations of pregnenolone, progesterone, allopregnanolone and allotetrahydrodeoxycorticosterone (THDOC) showed maximal increases in 96+/-3, 126+/-14, 110+/-12 and 70+/-13% above control, respectively, 30 to 60 min after injection of CB 34 (25 mg kg(-1)). CB 34 also increased the brain concentrations of neuroactive steroids in adrenalectomized-orchiectomized rats, although to a lesser extent than in sham-operated animals, suggesting that CB compounds stimulate brain steroidogenesis independently of their effects on peripheral tissues. The increase in brain and plasma neurosteroid content induced by CB 34 was associated with a marked anticonflict effect in the Vogel test. Our results indicate that the three CB compounds tested are specific and potent agonists at peripheral benzodiazepine receptors, and that they stimulate steroidogenesis in both the brain and periphery.

Benzodiazepines do not modulate desensitization of recombinant alpha1beta2gamma2 GABA(A) receptors.

Previous studies suggest that diazepam (DZP) increases the desensitization rate of GABA(A) receptors, although this effect could simply be a consequence of the DZP-induced increase in GABA sensitivity rather than a direct modulation of desensitization kinetics. To distinguish these two possibilities, voltage clamp recordings were performed on rat alpha1beta2gamma2 GABA(A) receptors expressed in Xenopus laevis oocytes. Complete GABA concentration-response relationships were obtained in the absence and presence of 1 microM DZP and the observed shift in GABA sensitivity (approximately 2.5-fold) was used to adjust GABA and GABA plus DZP to the same level of activation. In this case, DZP had no significant effect on either the rate of onset or recovery from desensitization. This suggests that the apparent effect of DZP on the rate of desensitization is secondary to the increase in GABA sensitivity and not due to a direct effect on the process of desensitization.

Clozapine's antipsychotic effects do not depend on blockade of 5-HT3 receptors.

Sixteen known 5-HT3 receptor blockers, including clozapine, fully or partially reverse the inhibitory effect of 1 microM GABA on [35S]TBPS binding, indicating that they are also GABA(A) antagonists, some of them selective for subsets of GABA(A) receptors. The 5-HT3 receptor blocker, ondansetron, has been reported to produce some antipsychotic and anxiolytic effects. However, no antipsychotic effects have been reported for a large number of highly potent 5-HT3 receptor blockers. Like clozapine, ondansetron partially reverses the inhibitory effect of GABA on [35S]TBPS binding. Additivity experiments suggest that ten 5-HT3 receptor blockers tested at low concentrations preferentially block subtypes of GABA(A) receptors that are among those blocked by clozapine. Wiley and Porter (29) reported that MDL-72222, the most potent GABA(A) antagonist described here, partially generalizes (71%) with clozapine in rats trained to discriminate an interoceptive clozapine stimulus, but only at a dose that severely decreases responding. Tropisetron (ICS-205,930) exhibits both GABA-positive and GABA-negative effects. R-(+)-zacopride is 6-fold more potent than S-(-)-zacopride as a GABA(A) antagonist. We conclude that the observed antipsychotic and, possibly, anxiolytic effects of some 5-HT3 receptor blockers are due to selective antagonism of certain GABA(A) receptors, and not to blockade of 5-HT3 receptors. We speculate that the anxiolytic and sedative effects of clozapine and several other antipsychotic drugs may be due to selective blockade of alpha1beta2gamma2 GABA(A) receptors which are preferentially located on certain types of GABAergic interneurons (probably parvalbumin positive). Blockade of these receptors will increase the inhibitory output of these interneurons. So far, no highly potent GABA(A) antagonists with clozapine-like selectivity have been identified. Such compounds may exhibit improved clozapine-like antipsychotic activity.